The Role Of H3K27me3 And KDM6A/B In The Mouse Nuclear Transfer Embryos

Somatic cell nuclear transfer (SCNT) is a technique for cloning. The nucleus is removed from a healthy oocyte. This oocyte becomes the host for a nucleus that is transplanted from another cell, such as a cumulus cell. The reconstructed embryo can be used to generate embryonic stem cells with a genetic match to the nucleus donor (Therapeutic cloning), or can be implanted into a surrogate mother to create a cloned individual, such as Dolly the sheep (Reproductive cloning). Nuclear transfer, cell fusion, specific transcription factors and culture conditions can reprogram the somatic cells. However, only the somatic cell nuclear transfer technology can be reprogrammed the cells to an animal, and the other method has some deficiencies. Since the establishment of somatic cell nuclear transfer technology, cloning efficiency at a relatively low level, only a few of the reconstructed embryos can develop to term birth, usually only 1-2% birth rate in mice, and most of the cloned mice will die soon after birth. Cloned mice often appear placenta, fetal growth and other abnormal symptoms, and this phenomenon is referred to as fetal giant. The main reason for this phenomenon is that the donor cell cannot be fully reprogrammed. Reprogramming mainly refers to the erase and rebuild of epigenetic modifications, including DNA methylation, histone acetylation, genomic imprinting, and X chromosome reconstruction reactivation, et al.KDM6A and KDM6B were found in 2007, and the related studies mainly focused on the biochemistry, the role of KDM6A/B in nuclear transfer has not been reported. The current study shows that other histone demethylase play an important role in the process of embryo development.In this study, we established a set of feasible methods for mouse cloning. By comparing the cumulus cell reconstructed embryo, fetal fibroblasts cell reconstructed embryo, and embryonic stem cell reconstructed embryo in different activation medium. Then the development efficiency and embryo transfer methods in different culture medium were compared. The results showed that 1) The KSOM Ca2+-free is a universal activation memedium, and the activation rate is about 93.5%; 2) KSOM-AA medium is the most suitable medium for cumulus cells, embryonic stem cell reconstructed embryo, however, the best in vitro culture medium for fetal fibroblast reconstructed embryos was aMEM medium; 3) The best method for embryo transfer is 2-cell stage oviduct transfer.We determined that the global levels of the H3K27me3 are elevated in MⅡ oocyte chromatin and decrease to minimal levels at the time of the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, KDM6A and KDM6B, are observed at high transcript and protein levels in mouse preimplantation embryos. In activated oocytes, when the H3K27me3 disappears at the 8-cell stage, the KDM6A (but not KDM6B) protein levels are undetectable. Using RNA interference, we suppressed KDM6A and KDM6B gene expression in the embryo and determined that the functions of KDM6A and KDM6B are complementary. When KDM6B levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of KDM6A produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.